A novel approach is proposed to develop a cholera subunit vaccine based on the current understanding of Vibrio cholerae (Vc) colonization, pathogenesis, and the human immune response to cholera. Currently, a killed, whole-cell (W-C) Vc vaccine delivered orally is used in much of the world where cholera is problematic. The degree of protection it induces depends on the age and the previous exposure of the vaccinees to Vc. The killed W-C cholera vaccines do not express immunogenic levels of several Vc protective antigens that are expressed during infection. To circumvent this, we propose a strategy based in part on the highly successful H. influenzae type b and B. pertussis vaccines that utilize either carbohydrate epitopes bound to carrier proteins, or multiple virulence factors and a toxoid to achieve long-lasting immunity. Individual antibodies (Abs) to Vc LPS and 3 colonization factors are protective in animal models. These Vc virulence factors will be used to configure a cholera subunit vaccine to induce a concentrated immune response to the critical first step(s) in Vc pathogenesis. We hypothesize that a subunit cholera vaccine, composed of 4 protective Vc colonization/adhesion factors will induce protective immunity with 1 dose. Detoxified (det) Inaba LPS will be conjugated to one of three virulence factors: Toxin Co-Regulated pilus A (TcpA), TcpF or chitin-binding protein A (CBP-A). Carrier-specific T cell help will enhance the anti-LPS response and help for induction of protective carrier-specific Abs. The studies bring together Drs. Wade and Grandjean who have extensive experience in cholera research. Dr. W. Wade is trained in immunopathogenesis. He pioneered the development of the vaccination protocols for synthetic Vc LPS epitopes conjugated to carrier proteins. He is an expert in the anti-Vc LPS response. Dr. Grandjean is an accomplished carbohydrate chemist who has generated Vc LPS:protein conjugates. Public Health Relevance: Cholera is still a disease that sickens millions and kills thousands yearly. We propose to formulate a new subunit cholera vaccine based on identification of protective epitopes in a known virulence proteins of V. cholerae: TcpA. Previous work from our lab has shown that LPS and TcpA can form the basis for a cholera vaccine. We will pursue the development of a cholera subunit vaccine based on detoxified-LPS bound to 3 protein component required for colonization of the strain of V. cholera that is causing the current pandemic.